POLYIMIDAZOLE CONJUGATED OLIGONUCLEOTIDES REACH THE NUCLEUS OF HELA CELLS

Oligonucleotide models bearing 6, 12 or 18 histamine residues were synthesized on solid support and labeled with fluorescein. Only the oligo with 6 histamine residues showed a high uptake in HeLa cells with a nuclear localization. Experiment a 4°C or with bafilomicyn A₁ suggest that uptake proceeded by an endocytosis mechanism followed by a destabilization of the membrane. Once in the cytoplasm the oligo reached rapidly the nucleus.     

Identification of oligothymidylates as new simple substrates for Escherichia coli DNA photolyase and their use in a rapid spectrophotometric enzyme assay

Escherichia coli DNA photolyase exhibits the same turnover number (3.4 min-1) for the repair of dimers in oligothymidylates [oligo(dT)n] containing 4-18 thymine residues. This rate is identical with that observed with polythymidylate and with native DNA. The enzyme exhibits a similar high affinity with oligomers containing seven or more thymine residues. A decrease in affinity is detectable with oligo(dT)n when n = 4-6. The enzyme is active with oligo(dT)3, but no evidence for saturation was obtained at dimer concentrations up to 15 microM where the observed repair rate is 43% of the turnover number observed with the higher homologues. Nearly quantitative (90-100%) repair is observed with oligo(dT)n when n is greater than or equal to 9. Photolyase can repair internal dimers and dimers at a 5' end where the terminal ribose is phosphorylated but not at unphosphorylated 5' or 3' ends. The latter can explain a progressive decrease in the extent of repair observed with short-chain oligomers. The observed specificity can also explain why the enzyme is inactive with oligo(dT)2 [p(dT)2] since the only dimer possible in oligo(dT)2 involves an unphosphorylated 3' end. That the enzyme can repair dimers in short-chain, single-stranded analogues for DNA suggests that in catalysis with DNA recognition of the dimer itself is important as opposed to recognition of the deformation in DNA structure produced by the dimer. Dimer repair with oligo(dT)n is detected by the increase in absorbance at 260 nm, a feature which is used as the basis for a rapid spectrophotometric assay with a lower detection limit around 150 pmol of dimer repaired.     

Studies on the specificity of uptake and release of radiolabelled histamine in rat brain slices

Previously it has been shown that radiolabelled histamine is taken up by brain slices and may subsequently be released by depolarizing stimuli in a calcium-dependent manner, indicating the involvement of neurons in uptake and release of histamine.The present study demonstrates that after incubation of brain slices with low (nM) concentrations of [³H]histamine the amine may be taken up by (and released from) dopaminergic and serotonergic neurons (nerve terminals). Thus 6-hydroxydopamine- and 5,7-dihydroxytryptamine-induced lesions not only reduced the uptake of [³H]dopamine (in striatal slices) and [³H]serotonin (in hippocampal slices), but also, though to a lesser extent, that of [³H]histamine. Immunocytochemical findings revealed that the neurotoxins did not visibly affect histaminergic neurons. Lesioning of noradrenergic neurons appeared not to alter significantly the uptake of [³H]histamine. Further, various drugs acting on either catecholamine-, serotonin- or opioid-receptors and known to cause presynaptic inhibition of the release of [³H]dopamine or [³H]wrotonin from striatal or hippocampal slices also inhibited [³H]histamine release.It is concluded that incubation of brain slices with low concentrations of [³H]histamine does not result in a selective labelling of histaminergic neurons. The possibility that, unlike other monoamines, histamine is not subject to high-affinity uptake by the nerve terminals from which it was released, is discussed.     

Histamine stimulated synaptosomal Ca2+ uptake through activation of calcium channels

Histamine stimulated Ca2+ uptake in synaptosomes was completely inhibited by the slow Ca2+ channel antagonists verapamil, cinnarizine and flunarizine, and slightly inhibited by nifedipine and diltiazem. Ca2+ uptake in synaptosomes depolarized or predepolarized with varying K+ concentrations was increased by histamine, in both conditions, until 30mM K+. At higher K+ concentrations histamine was not able to alter K+ effects in either conditions. 30mM K+ stimulated uptake of Ca2+ in the absence or presence of histamine was not inhibited by verapamil and diltiazem. However nifedipine slightly inhibited K+ and K+ +histamine effects. 3-Isobutyl-1-methyl-xanthine and dibutyryl cyclicAMP potentiated (10%) the uptake of Ca2+ in synaptosomes induced by histamine. Dibutyryl cyclicAMP alone however decreased the basal Ca2+ uptake in a concentration-dependent manner. Verapamil, but not diltiazem, antagonized the effects elicited by 3-isobutyl-1-methyl-xanthine and dibutyryl cyclicAMP in the presence of histamine. The data suggest that the increase in synaptosomal Ca2+ uptake induced by histamine is mediated by the activation of the voltage sensitive calcium channels, and possibly a cyclicAMP-dependent protein kinase phosphorylation can modulate the opening of Ca2+ channels.     

Probing cellular processes with oligo-mediated recombination and using the knowledge gained to optimize recombineering

Recombination with single-strand DNA oligonucleotides (oligos) in Escherichia coli is an efficient and rapid way to modify replicons in vivo. The generation of nucleotide alteration by oligo recombination provides novel assays for studying cellular processes. Single-strand exonucleases inhibit oligo recombination, and recombination is increased by mutating all four known exonucleases. Increasing oligo concentration or adding nonspecific carrier oligo titrates out the exonucleases. In a model for oligo recombination, λ Beta protein anneals the oligo to complementary single-strand DNA at the replication fork. Mismatches are created, and the methyl-directed mismatch repair (MMR) system acts to eliminate the mismatches inhibiting recombination. Three ways to evade MMR through oligo design include, in addition to the desired change (1) a C·C mismatch  6 bp from that change; (2) four or more adjacent mismatches; or (3) mismatches at four or more consecutive wobble positions. The latter proves useful for making high-frequency changes that alter only the target amino acid sequence and even allows modification of essential genes. Efficient uptake of DNA is important for oligo-mediated recombination. Uptake of oligos or plasmids is dependent on media and is 10,000-fold reduced for cells grown in minimal versus rich medium. Genomewide engineering technologies utilizing recombineering will benefit from both optimized recombination frequencies and a greater understanding of how biological processes such as DNA replication and cell division impact recombinants formed at multiple chromosomal loci. Recombination events at multiple loci in individual cells are described here.     

A method for isolation of oligo(uridylic acid)-containing messenger ribonucleic acid from HeLa cells

When cytoplasmic polyadenylated ribonucleic acid [poly(A+)RNA] from HeLa cells was treated with ribonuclease H (RNase H) and oligodeoxythymidylate [oligo(dT)] to remove its 3'-poly(A) tail, an increased binding to poly(A)-agarose was observed. The bound material, which comprised 4-6% of the initial RNA, contained 65-80% of the oligo(uridylic acid) [oligo(U)] sequences generated by RNase T1 digestion. Oligo(U) isolated from the bound fraction was shown to be 83% U and to have a U/G ratio of 33. In contrast, oligo(U) from the unbound material was 77% U and had a U/G ratio of 13, suggesting that it is shorter and less U rich than the oligo(U) in the bound fraction. On sucrose gradients, oligo(U+)RNA consistently sedimented with a larger s value than oligo(U-) RNA. The oligo(U) content of oligo(U+) RNA suggests one oligo(U) tract of 33 nucleotides per RNA molecule of 2000-3000 residues.     

Aminopyrine uptake by guinea pig gastric mucosal cells. Mediation by cyclic AMP and interactions among secretagogues

The role of cyclic nucleotides in regulating acid secretion by dispersed mucosal cells from guinea-pig stomach was examined by measuring first the ability of histamine and carbachol to stimulate [dimethylamine-14C]aminopyrine uptake and cyclic nucleotide metabolism and secondly, the effect of exogenous cyclic nucleotides on basal and stimulated [14C]aminopyrine uptake. The [14C]aminopyrine was found in an acidic, osmotically sensitive compartment, probably associated with the initial steps in acid secretion by these cells. Although histamine increased [14C]aminopyrine uptake and cyclic AMP synthesis as expected, histamine was approx. 10-fold more potent in inducing [14C]aminopyrine uptake. This dissociation of [14C]aminopyrine uptake and cyclic AMP metabolism process was further manifested by the observation that prostaglandin E1 failed to increase [14C]aminopyrine uptake, although it did cause a rise in cellular cyclic AMP. Furthermore, prostaglandin E1 did not alter the [14C]-aminopyrine uptake caused by histamine. Carbachol was found to increase the [14C]aminopyrine uptake and also to potentiate the ability of histamine to increase [14C]aminopyrine uptake. Carbachol, however, affected neither the histamine-induced increase in cyclic AMP nor the binding of [3H]histamine to the cells. Cimetidine, a histamine H2 receptor antagonist, blocked the [14C]aminopyrine uptake induced either by histamine alone or by the potentiating combination of histamine plus carbachol. These results suggest that cyclic AMP is mediating the action of histamine on [14C]aminopyrine uptake but changes in cyclic AMP per se are not necessarily the cause for the potentiated increase in [14C]aminopyrine uptake. Furthermore, the potentiated response observed with histamine plus carbachol on [14C]aminopyrine uptake occurs at a biochemical step distal to and not obviously related to cyclic AMP generation.     

Quantitative determination of poly(adenosine diphosphate ribose) in different hepatic tissues by an isotope dilution procedure.

A procedure has been developed for the quantitation of poly(ADP-ribose) in intact tissues. It is based on the dilution of added [3H]poly(ADP-ribose) by the endogenous polymer. 5 - 6 nanomoles protein-bound ADP-ribose per mg DNA were found in adult and neonatal rat liver, while Zajdela hepatoma cells had significantly lower values. A comparison with mono(ADP-ribose) residues in adult rat liver revealed similar levels of monomeric and polymeric ADP-ribose residues. This means that far more proteins (or acceptor sites on proteins) must be occupied by single ADP-ribose residues than by oligo or poly(ADP-ribose) chains. While the poly(ADP-ribose) levels of the different tissues do not correlate with the corresponding proliferation rates, the amount of mono(ADP-ribose) does show a certain Correlation, being low in rapidly growing tissues.     

Aggregates of oligo(dG) bind and inhibit topoisomerase II activity and induce formation of large networks.

DNA cleavage by eukaryotic type II DNA topoisomerase (EC 5.99.1.3) was strongly inhibited by an oligonucleotide containing 10 dGua residues. Catalytic activities of topoisomerase II, as measured by relaxation and decatenation reactions, were also inhibited by oligo(dG)10. Inhibition was specific to oligo(dG)10; other oligonucleotides, nucleotides, or single-stranded DNAs tested did not influence the activity of topoisomerase II. Oligo(dG)10 did not inhibit other activities such as restriction enzymes. Although the enzyme neither binds nor cleaves oligo(dG)10, inhibition can be explained by the finding that topoisomerase II binds tightly with aggregated oligo(dG) structures (estimated to contain between 20 and 30 molecules of monomeric oligo(dG)10) that form spontaneously prior to addition of enzyme. These aggregated oligo(dG)-topoisomerase complexes are large networks that can be pelleted by a 20-min centrifugation step in a Microfuge. Western blotting with a monoclonal antibody confirmed that topoisomerase II is trapped in these pellets. The ability of the enzyme to form large DNA-protein networks could be a biochemical mechanism by which topoisomerase II might promote or participate in chromosome condensation in vivo prior to mitosis.     

Effects of hormones (calcitonin, GIP) and pharmacological antagonists (ranitidine and famotidine) on isolated rat parietal cells

The rationale for the present study was to compare calcitonin and gastric inhibitory polypeptide (GIP) versus two histamine H2 receptor antagonists with respect to their potency of inhibiting parietal cell functions. Adenylate cyclase activity and acid production ([14C]aminopyrine uptake) of isolated rat parietal cells were stimulated by histamine. At 10(-7) and 10(-6) mol/l, calcitonin and GIP reduced the response to histamine by 10-20% following noncompetitive kinetics. Ranitidine and famotidine (MK 208) inhibited the response to histamine by about 50% at 10(-7)-10(-6) mol/l, and at 10(-5) mol/l abolished the histamine effect. On a molar basis famotidine turned out to be 6 times more potent than ranitidine. Both antagonists revealed competitive kinetics. Our data suggest direct inhibition of the parietal cells by the tested compounds which were shown to interfere at the adenylate cyclase cAMP system or at the histamine H2 receptor. However, compared to the histamine H2 receptor antagonists, hormonal inhibition is less pronounced and mediated by a different mechanism.     

Regional Characteristics of Histamine Uptake into Neonatal Rat Astrocytes

Histaminergic signalling constitutes an attractive target for treatment of neuropsychiatric disorders. One obstacle to developing new pharmacological options has been failure to identify putative specific histamine transporter responsible for histamine clearance. Although high-affinity histamine uptake was detected in neonatal cortical astrocytes, its existence in other brain regions remains largely unexplored. We investigated whether cerebellar and striatal astrocytes participate in histamine clearance and evaluated the role of organic cation transporters (OCT) in astroglial histamine transport. Kinetic and pharmacological characteristics of histamine transport were determined in cultured astrocytes derived from neonatal rat cerebellum, striatum and cerebral cortex. As well as astrocytes of cortical origin, cultured striatal and cerebellar astrocytes displayed temperature-sensitive, high-affinity histamine uptake. Exposure to ouabain or Na⁺-free medium, supplemented with choline chloride markedly depressed histamine transport in cortical astrocytes. Conversely, histamine uptake in striatal and cortical astrocytes was ouabain-resistant and was only partially diminished during incubation in the absence of Na⁺. Also, histamine uptake remained unaltered upon exposure to OCT inhibitor corticosterone, although OCTs were expressed in cultured astrocytes. Finally, histamine transport in cerebellar and striatal astrocytes was not sensitive to antidepressants. Despite common characteristics, cerebellar astrocytes had lower affinity, but markedly higher transport capacity for histamine compared to striatal astrocytes. Collectively, we provide evidence to suggest that cerebellar, striatal as well as cortical astrocytes possess saturable histamine uptake systems, which are not operated by OCTs. In addition, our data indicate that Na⁺-independent histamine carrier predominates in cerebellar and striatal astrocytes, whereas Na⁺-dependent transporter underlies histamine uptake in cortical astrocytes. Our findings implicate a role for histamine transporters in regulation of extracellular histamine concentration in cerebellum and striatum. Inhibition of histamine uptake might represent a viable option to modulate histaminergic neurotransmission.     

基于模板切换的mRNA5'末端全长扩增方法

本文介绍一种称为CapFinder的技术,可用于克隆基因mRNA序列的5'末端非翻译区全长。该技术是利用某些反转录酶在反转录达到mRNA的5'末端帽结构时表现出很高的加尾活性(主要添加dC)这一特点,在反转录体系中加入一种带GGG的寡核苷酸序列,当反转录反应到达mRNA模板的5'末端帽结构时,切换到以该寡核酸为模板继续进行反转录反应,即可合成完整的cDNA一链,且在其3'末端还带有一段额外的寡核苷酸序列。用GGG寡核苷酸序列为上游引物和基因特异性的下游引物进行PCR即可扩增得到mRNA5'末端非翻译区的全长。利用该技术克隆了棉铃虫幼虫中肠Bt毒素受体E-钙粘素基因的5'末端非翻译区序列。     

Directed cleavage of ribopolynucleotides with nucleases restricted by multiple modification of substrate.

Simultaneous exhaustive modification of cytidine and uridine residues of rRNA with methoxyamine and sodium metabisulfite renders adjacent phosphodiester bonds resistant to pancreatic and T2 ribonucleases. Another method of T2 RNAase restriction is modification of cytidine with methoxyaminebisulfite followed by modification of guanosine residues with beta-ethoxy-alpha-ketobutyraldehyde. Mild alkaline treatment leads to demodification of uridine and guanosine residues leaving intact modified cytidine residues, thus providing a means of stepwise, directed cleavage of the polynucleotide. The series of combined cleavage procedures and methods of isolation of oligo(C), oligo(G) and oligopyrimidine tracts, as well as the procedure of selective cleavage at uridine residues elaborated in the course of the present studies may serve as a basis for more rational procedures of RNA sequencing.     

The effect of platelet-activating factor on [14C]aminopyrine uptake by isolated guinea pig parietal cells

C16- and C18- platelet-activating factor (PAF) at 10(-6) M enhanced the uptake of [14C]aminopyrine (AP) by isolated guinea pig parietal cells. This increase was inhibited by a PAF antagonist CV-6209. In a medium with a low calcium (Ca2+) concentration (2 uM), this increase was not observed, which indicates that the increase of AP uptake by PAF is dependent on extracellular Ca2+. PAF and lyso-PAF showed no effect on AP uptake in the presence of histamine or carbachol.     

Interaction of Escherichia coli host factor protein with oligoriboadenylates

The interaction of Escherichia coli host factor 1 with oligoadenylate [oligo(A)] was studied by fluorescence and filter retention techniques. The intrinsic fluorescence of the host factor is quenched by up to 60% by the addition of oligo(A). Fluorescence titrations at high protein concentrations (6 microM) give a saturation point of 14 A residues per host factor hexamer regardless of chain length or ionic strength. Nitrocellulose filter retention experiments at much lower concentrations (1 nM) indicate equimolar complexes form between (pA)l (12 less than l less than 27) and host factor hexamers. The smallest number of contiguous A residues which allows the formation of all favorable protein--RNA contacts is 16 at both low and high salt concentrations. At 0.1 M NaCl, the molar association constants are in the range of 10(10)--10(11) M-1 (15 less than l less than 27) and decrease only slightly with ionic strength, indicating a large nonionic component in the interaction. Cyclized (pA)l was shown to have a higher affinity for host factor than its linear counterparts when l is 18 or greater but a lower relative affinity when l is 15. This suggests that the binding site on the hexamer has a circular spatial orientation.     

Histamine (re)uptake by astrocytes: an experimental and computational study

Astrocytes participate in the clearance of neurotransmitters by their uptake and subsequent enzymatic degradation. Histamine as a polar and/or protonated molecule must use a carrier to be transported across the cell membrane, although a specific histamine transporter has not been elucidated, yet. In this work we upgraded the kinetic studies of histamine uptake into neonatal rat cultured type 1 astrocytes with quantum chemical calculations of histamine pKa values in conjunction with Langevin dipoles solvation model as the first step toward microscopic simulation of transport. Our results indicate that astrocytes transport histamine by at least two carrier mediated processes, a concentration gradient dependent passive and a sodium-dependent and ATP-driven active transport. We also demonstrated that histamine protonation states depend on the polarity of the environment. In conclusion we suggest that histamine, a polar molecule at physiological pH uses at least two different mechanisms for its uptake into astrocytes –an electrodiffusion and Na⁺-dependent and ouabain sensitive active process. We emphasize relevance of knowledge of histamines protonation states at the rate limiting step of its transport for microscopic simulation that will be possible when structure of histamine transporter is known.     

Histamine secretion from permeabilized mast cells by calcium

A transient increase in the permeability of the mast cell membrane was caused by the exposure of the cells to low concentrations of saponin, 5 or 10 micrograms/ml. These concentrations had very little effect in the absence of calcium but caused 35 to 50% histamine release, having the character of a secretory response, when 0.25 mM or more calcium was added to the medium. The dose-response curve was steep between 25 microM and 250 microM calcium and tended to flatten with higher concentrations. The release was associated with a pronounced increase in calcium uptake, which was faster than the histamine release. The membrane changes were slight as indicated by only 7 to 12% leakage of lactate dehydrogenase and by the absence of any detectable change in the electron micrographs. The transient nature of the membrane change is shown by the following experiment. When the cells were first exposed to saponin in the absence of calcium, the amount of histamine released by the subsequent incubation with calcium varied inversely with the time interval that elapsed before calcium was added. If calcium was added after 15 minutes no histamine release occurred. When calcium uptake was studied in the same manner, the stimulation of calcium uptake in saponin-treated cells also declined progressively with increasing intervals after the exposure to saponin when calcium was added. Stimulation of both histamine release and calcium uptake was inhibited by antimycin A, the inhibition curves with 10(-9)M to 10(-7)M antimycin A being similar. The effect on the calcium uptake by itself could explain the inhibition of histamine release. But the release was also inhibited by the calmodulin antagonists, W-7 and mepacrine, suggesting that the influx of calcium in the permeabilized cells acts primarily through calmodulin-mediated enzyme activation.     

Flow cytometry and live confocal analysis for the evaluation of the uptake and intracellular distribution of FITC-ODN into HaCaT cells

In this study, the mechanism of the internalization and the cellular distribution of 59 fluorescein conjugated PS-ODN (FITC-ODN) after transfection with different mixed lipidic vesicles/oligo complexes (lipoplexes) have been investigated. Mixed lipidic vesicles were prepared with one of the most used cationic lipid (DOTAP) and different amounts of a cholic acid (UDCA) to release the oligo into HaCaT cells. Using flow cytometry, the cellular uptake of the oligo was studied with and without different inhibitors able to block selectively the different pathways involved in the internalization mechanism. The intracellular distribution of the oligo was analyzed by confocal laser scanning microscopy (CLSM), treating the cells with the lipoplexes and directly observing without any fixing procedure. To better carry out the colocalization studies, fluorescent-labeled markers, specific for the different cellular compartments, were coincubated with 59 fluorescein-conjugated 29-mer phosphorotioate oligonucleotide (FITC-ODN). The different lipidic vesicles affect the internalization mechanism of FITC-ODN. After using the inhibitors, the uptake of complexes involved a different internalization mechanism. The live CLSM analysis demonstrated that, after 1 hour from the complex incubation, the oligo was transferred into cells and localized into the endosomes; after 24 hours, the oligo was intracellularly localized close to the nuclear structure in a punctuate pattern. However, the results from fusion experiments showed also a binding of a quite low amount of oligo with the cell membranes.